This project was initiated to analyze, both qualitatively and quantitatively, changes in total cellular protein patterns during cellular differentiation using the technique of quantitative two-dimenional electrophoresis. Results obtained to date include: (1) mouse epidermal cells were cultured in the presence of either low calcium (0.02-0.1 mM) (mainly basal cells) or high calcium (1.2 mM)(mainly mature keratinocytes) and then treated with l2-0-tetradecanoylphorbol-l3-acetate (TPA) for 1, 4, or 24 hours. Two-dimensional analysis of total cellular polypeptides from cells grown in the presence of either low or high calcium revealed only quantitative polypeptide differences. (2) Similarly, only quantitative differences were detected following treatment of either low or high calcium cells with TPA (0.01 and 0.1 microgram/ml). In low calcium cells, roughly 3 and 6%, respectively, of the total number of polypeptides (800 paired spots) from TPA-treated cells underwent quantitative changes greater than four-fold. In high calcium cells, however, TPA treatment had a much lesser effect on gene expression. Only 4/854 (0.5%) and 15/934 (1.6%) of the total number of polypeptides changed greater than four-fold following TPA treatment. (3) Comparison of the common changing polypeptides that were observed following treatment with either TPA (0.1 microgram/ml) or during exposure of cells to high calcium revealed 16 common changing polypeptides (four-fold differences). In 14/16 of the polypeptides the common changes were also in the same direction (e.g., if the polypeptide increased following treatment with TPA, the same polypeptide also increased in the presence of high calcium and vice versa). Eight polypeptides were increased following treatment with TPA or growth in high calcium and six decreased following treatment.